The genomes of many positive sense RNA viruses contain an internal ribosomal entry site (IRES) that mediates end-independent initiation of translation. IRESs belong to different structural classes and use distinct mechanisms for initiation. Our proposed studies of the process of initiation on representatives of four classes of IRES that use distinct mechanisms will identify important cis-acting elements and provide detailed mechanistic insights into the actions of canonical initiation factors and cellular IRES trans-acting factors (ITAFs) in promoting internal ribosomal entry. Our studies will address mechanisms of increasing complexity. The ~180 nt-long intergenomic region (IGR) IRESs of dicistroviruses mediate initiation without initiator tRNA or initiation factors and the first elongation cycle consequently occurs without deacylated tRNA in the ribosomal P site. We will characterize whether elements in the IRES play an analogous role during elongation to deacylated tRNA when it has been translocated to the E site. The IRES of classical swine fever virus, a pestivirus, is substantially resistant to inhibition by eIF2 phosphorylation, which can be accounted for by the use of two exceptional eIF2-independent mechanisms of initiation. We will determine whether these mechanisms account for the relaxation on this IRES of the ribosome's normally strict avoidance of initiation at non-AUG codons. Type 2 picornavirus IRESs such as the encephalomyocarditis virus IRES bind specifically to eIF4G/eIF4A: we will investigate how these factors promote recruitment of 43S complexes to the initiation codon, how ITAFs induce conformational changes in these IRESs and why only a subset of type 2 IRESs require their activity. The mechanism of initiation on type 1 picornaviruses (e.g. poliovirus) is not known, but also involves specific interaction with eIF4G. We shall characterize this interaction in detail, validate previously identified candidate ITAFs and if necessary isolate additional ITAFs to reconstitute the entire initiation process on this IRES in vitro. We will then characterize interactions between factors and the IRES using chemical/enzymatic footprinting and directed hydroxyl radical cleavage. The studies will provide a framework for understanding of mechanistic details of IRES-mediated initiation, for understanding the cell-type specificity of IRES function and for the design of inhibitors.